Underivatized, aqueous soluble xcex2(1-3)-glucan (also known as PGG-Glucan or Betafectin(copyright)) is a novel and unique soluble xcex2-glucan manufactured through a proprietary process. The biological activity of this molecule is clearly distinguishable from particulate or other soluble xcex2-glucans. Numerous laboratories have reported direct induction of arachidonic acid metabolites (Czop et al., J. Immunol. 141(9):3170-3176 (1988)), cytokines (Abel and Czop, Intl. J. Immunopharmacol. 14(8):1363-1373 (1992); Doita et al., J. Leuk. Biol. 14(2):173-183 (1991)) and oxidative burst (Cain et al., Complement, 4:75-86 (1987); Gallin et al., Int. J. Immunopharmacol. 14(2):173-183 (1992)) by both particulate and soluble forms of xcex2-glucans. In contrast, underivatized, aqueous soluble xcex2(1-3)-glucan does not directly activate leukocyte functions such as oxidative burst activity (Mackin et al., FASEB J. 8:A216 tokine secretion (Putsiaka et al., Blood 82:3695-3700 (1993)) or proliferation (Wakshull et al., J. Cell. Biochem. suppl. 18A:22 (1994)). Instead, underivatized, aqueous soluble xcex2(1-3)-glucan primes cells for activation by secondary stimuli (Mackin et al. (1994); Brunke-Reese and Mackin, FASEB J. 8:A488 (1994); and Wakshull et al. (1994)).
The biological activity of xcex2-glucans is mediated through specific receptors located on target cells. Several groups of investigators have described receptors which bind particulate xcex2-glucan preparations. For example, receptors for particulate xcex2-glucans (e.g., zymosan-like particles) have been described by Czop and colleagues (Czop and Kay, J. Exp. Med. 173:1511-1520 (1991); Szabo et al., J. Biol. Chem. 270:2145-2151 (1995)) and Goldman (Immunology 63(2):319-324 (1988); Exp. Cell. Res. 174(2):481-490 (1988)). The leukocyte complement receptor 3 (CR3, also known as MAC 1 or CD11b/CD18) has been shown to have the capacity to bind both particulate and some soluble xcex2-glucans, as well as other polysaccharides (Thornton et al., J. Immunol. 156:1235-1246 (1996)). A soluble aminated xcex2-glucan preparation has been shown to bind to murine peritoneal macrophages (Konopski et al., Biochim. Biophys. Acta 1221:61-65 (1994)), and a phosphorylated xcex2-glucan derivative has been reported to bind to monocyte cell lines (Muller et al., J. Immunol. 156:3418-3425 (1996)). The ability of salmon macrophages (Engstad and Robertsen, Dev. Comp. Immunol. 18(5):397-408 (1994)) and brain microglial cells (Muller et al., Res. Immunol. 145:267-275 (1994)) to phagocytose xcex2-glucan particles, presumably through a receptor-mediated process, has also been described.
Unfortunately, each group has utilized xcex2-glucan preparations varying widely in their source, method of preparation, purity and characterization. In addition, different cell types and species, both primary and established cell lines, and different functional read-outs have been used. The relationship between the various receptors described by these investigators has, therefore, not been defined, although it is clear that the receptor described by Czop is not CR3 (Szabo et al. (1995)).
This invention pertains to the discovery that underivatized, aqueous soluble xcex2(1-3)-glucan specifically binds to a novel receptor located on human leukocyte membranes (HLM). As described herein, a radiolabeled underivatized, aqueous soluble xcex2(1-3)-glucan was used to measure the binding of this xcex2-glucan to membrane receptors derived from human leukocytes as well as various murine and human cell lines. The receptor for underivatized, aqueous soluble xcex2(1-3)-glucan shows specific and saturable binding to membranes and is highly selective for a subclass of soluble xcex2-glucans. Results of work described herein characterize this receptor for underivatized, aqueous soluble xcex2(1-3)-glucan and clearly differentiate it from previously described xcex2-glucan receptors for either particulate or soluble xcex2-glucans, while revealing important information about the mechanism of underivatized, aqueous soluble xcex2(1-3)-glucan biological activity.
This invention also pertains to a method of altering (e.g., activating or deactivating) signal transduction pathways, for example through modulation of one or more transcriptional regulatory factors in receptor-positive cells, i.e., cells which contain the receptor for underivatized, aqueous soluble xcex2(1-3)-glucan. In one embodiment of the invention, the signal transduction pathway is modulated or regulated by one or more transcriptional regulatory factors from the NF-xcexaB and/or NF-IL6 and/or jun/fos families of transcriptional regulatory factors. For example, the transcriptional regulatory factor can be NF-xcexaB, NF-IL6 or AP-1.
Other signal transduction pathways which can be altered by the methods of the present invention include the ras/raf-1/MAP kinase pathway, the G-protein/phospholipase C/protein kinase C pathway, the JAK/STAT pathway, the phospholipase A pathway, G-protein/phospholipase D/phosphatidic acid pathway and the c-AMP-dependent pathway. In each pathway, an appropriate activator or indicator of the signal pathway is activated by binding of underivatized, aqueous soluble xcex2(1-3)-glucan to its receptor, and modulation of this binding can alter the corresponding signal transduction.
According to the method of the present invention, the activity of the receptor for underivatized, aqueous soluble xcex2(1-3)-glucan is activated through binding of an underivatized, aqueous soluble xcex2(1-3)-glucan, whereby a signal transduction process is activated such that one or more transcriptional regulatory factors (e.g., from the NF-xcexaB, NF-IL6 or jun/fos families) are activated. Activation of these transcriptional regulatory factors can be used to measure the activation of the associated signal transduction pathway. Activation of the receptor can comprise, among others, an alteration in the receptor conformation, formation of a ligand-receptor complex, or alteration of the ligand-receptor complex. Alternatively, the activity of the receptor can be initiated by an agent which mimics the binding and activation ability of an underivatized, aqueous soluble xcex2(1-3)-glucan. In a particular embodiment, the transcriptional regulatory factor is activated as a result of ligand binding. In another embodiment, the activity of the transcriptional regulatory factor is decreased, either partially or totally, by the binding of an agent to the receptor (and thus excludes the underivatized, aqueous soluble xcex2(1-3)-glucan), but lacks the ability to activate the receptor.
The invention also pertains to an assay for identifying agents which alter (e.g., increase or decrease) the binding of underivatized, aqueous soluble xcex2(1-3)-glucan to its receptor. The assay comprises combining radiolabeled underivatized, aqueous soluble xcex2(1-3)-glucan, the receptor for underivatized, aqueous soluble xcex2(1-3)-glucan, and an agent to be tested, under conditions suitable for binding of underivatized, aqueous soluble xcex2(1-3)-glucan to its receptor. The extent of binding of underivatized, aqueous soluble xcex2(1-3)-glucan to its receptor in the presence of the agent to be tested is determined and compared with the extent of binding in the absence of the agent to be tested; a difference in the extent of binding indicates that the agent alters the binding of underivatized, aqueous soluble xcex2(1-3)-glucan to its receptor. An increase in the extent of binding in the presence of the agent indicates that the agent enhances, i.e., prolongs or increases, binding or is an agonist of the receptor for underivatized, aqueous soluble xcex2(1-3)-glucan. A decrease in the extent of binding in the presence of the agent indicates that the agent diminishes, i.e., shortens or decreases, binding or is an antagonist of the receptor for underivatized, aqueous soluble xcex2(1-3)-glucan. The invention also relates to agents identified by assays described herein, and accordingly, relates to agonists and antagonists of underivatized, aqueous soluble xcex2(1-3)-glucan activity.
The present invention also pertains to a novel assay for identifying agents which alter (e.g., increase or decrease) the effect of underivatized, aqueous soluble xcex2(1-3)-glucan on cellular signal transduction pathways, such as activation of transcriptional regulatory factors. This assay comprises combining underivatized, aqueous soluble xcex2(1-3)-glucan, the receptor for underivatized, aqueous soluble xcex2(1-3)-glucan and an agent to be tested under conditions in which binding of underivatized, aqueous soluble xcex2(1-3)-glucan to its receptor occurs (i.e., conditions suitable for binding of underivatized, aqueous soluble xcex2(1-3)-glucan to the receptor for underivatized, aqueous soluble xcex2(1-3)-glucan). Binding of underivatized, aqueous soluble xcex2(1-3)-glucan to its receptor activates the receptor, which in turn activates a signal transduction as exemplified or measured by a modulation of one or more transcriptional regulatory factors such as those from the NF-xcexaB, NF-IL6 or jun/fos families. The extent of activation of the selected transcriptional regulatory factor in the presence of an agent to be tested is determined and compared with the extent of activation of the selected transcriptional regulatory factor in the absence of the agent to be tested; a difference in the extent of activation indicates that the agent alters the effect of underivatized, aqueous soluble xcex2(1-3)-glucan on activation of the transcriptional regulatory factor. An increase in the activation of the transcriptional regulatory factor in the presence of the agent indicates that the agent enhances, i.e., prolongs or increases, the activation. A decrease in the activation of the transcriptional regulatory factor in the presence of the agent indicates that the agent diminishes, i.e., shortens or decreases, the activation.
The assays and methods of the present invention can be used to identify agents and drugs for use in treatment of infectious disease, inflammation, autoimmune diseases, ischemia reperfusion injury, cancer, asthma and hypersensitivity disorders. The assays and methods described herein can also be used to identify drugs which prolong the underivatized, aqueous soluble xcex2(1-3)-glucan effect, and therefore can be used in any therapeutic or prophylactic application in which underivatized, aqueous soluble xcex2(1-3)-glucan can be used, such as for immunomodulation, hematopoiesis, prevention and treatment of infectious disease, platelet production, peripheral blood precursor cell mobilization and wound healing. These agents or drugs act to enhance the effects of underivatized, aqueous soluble xcex2(1-3)-glucan by, for example, prolonging the binding of the glucan to its receptor or the effects thereof.
The present invention also relates to agents or drugs, such as, but not limited to, peptides or small organic molecules designed with reference to the binding site for underivatized, aqueous soluble xcex2(1-3)-glucan on the receptor for underivatized, aqueous soluble xcex2(1-3)-glucan. In one embodiment, such agents or drugs can be designed to mimic the activity of the receptor binding site in that they bind underivatized, aqueous soluble xcex2(1-3)-glucan, thus decreasing the amount of the xcex2(1-3)-glucan which is available for binding to the receptor and decreasing the activation of downstream events such as signal transduction. The present invention also pertains to an agonist or mimic of underivatized, aqueous soluble xcex2(1-3)-glucan activity with respect to its binding and activation of the receptor for underivatized, aqueous soluble xcex2(1-3)-glucan. Alternatively, the drug or agent can be designed to bind the receptor binding site, rendering it unavailable for binding by underivatized, aqueous soluble xcex2(1-3)-glucan; the present invention also relates to antagonists of underivatized, aqueous soluble xcex2(1-3)-glucan binding activity.
The work described herein has application to many areas. For example, it can be used in the monitoring of the underivatized, aqueous soluble xcex2(1-3)-glucan manufacturing process and product characterization for commercial release, to measure xcex2-glucans in fluids, to assess and determine structure-activity relationships of agents that interact with the receptor for the underivatized aqueous soluble xcex2(1-3)-glucan. Additionally, this work has application to the targeted delivery of various agents, including drugs and small molecules, to receptor-positive cells such as peripheral polymorphonuclear leukocytes, monocytes, macrophages and epithelial cells. The results described herein can also be used in purification schemes to enrich for both receptor-positive cells and receptor-negative cells, as well as in the generation of anti-receptor antibodies for diagnostic purposes.